Process for producing methylcobalamin

ABSTRACT

The present invention provides a novel industrially excellent and ecological process for producing methylcobalamin which is useful as a medicament etc. More specifically, it provides a process for producing methylcobalamin by reducing cyanocobalamin or hydroxocobalamin in the presence of a reducing agent, and then methylating the reductant by adding a water-soluble methylating agent.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP02/05510 which has an Internationalfiling date of Jun. 4, 2002, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to an industrially excellent process forproducing methylcobalamin. Specifically, it also relates to a novelproduction method which is free from the formation of a malodorousharmful substance and is ecological, and to a process for inhibiting theformation of such a malodorous harmful substance in a production processof methylcobalamin (V).

PRIOR ART

Methylcobalamin is a coenzymatic vitamin B12 present in the blood andspinal fluid. It can more satisfactorily migrate into the nerve tissuesthan the other B12 homologues and is used for prophylaxis, therapy, andamelioration of diabetic neuropathy, multiple neuritis, and otherperipheral neuropathy, especially of numbness, pain, and paralysis andfor megaloblast anaemia caused by a deficiency of vitamin B12.

Methylcobalamin has been conventionally produced by the followingpreparation methods:

-   (1) a method of reacting hydroxocobalamin with a dicarboxylic acid    monomethyl ester in the presence of a powdered metal (JP-A    49-47899);-   (2) a method of reacting cyanocobalamin with monomethyl oxalate in    hydrous methanol in the presence of a powdered metal (JP-A    50-41900);-   (3) a method of reacting hydroxocobalamin with methylmercury iodide    or ammonium methylhexafluorosilicate (JP-A 50-38120); and-   (4) a method of reacting cyanocobalamin with methyl iodide in the    presence of sodium borohydride (JP-A 45-38059).

However, dicarboxylic acid monomethyl esters such as monomethyl oxalateused in the methods (1) and (2) are not commercially available, must beprepared before use and cannot be used in commercial production. Inaddition, zinc powder used as the powdered metal is a heavy metal, it isindispensable to take measures for preventing its contamination intoproducts and for protecting the environment, and it is not industriallydesirable.

In the method (3), methylmercury iodide used is a pollutant and cannotbe used industrially. In addition, ammonium methylhexafluorosilicate isnot commercially available, must be prepared before use cannot be usedindustrially.

In contrast, the production method (4) is very excellent in view ofyield and product purity. However, methyl iodide has a very low boilingpoint (41° C. to 43° C.) and is thereby difficult to handle.Accordingly, this method is not sufficient as an industrial method forcommercial production. In addition, from the viewpoint of protectingworking environment or natural environment, the use of methyl iodideassigned as a specified chemical substance and having toxicity such aspotential carcinogenicity is by no means preferable in view ofindustrial health of factory workers. To obtain highly puremethylcobalamin by the method using methyl iodide, one or morepurification procedures by column chromatography are generallynecessary, thus inviting a serious problem in operation and productioncost. In addition, the column purification requires a large amount of anorganic solvent and an enormous quantity of a waste liquid.

Thus, an industrially excellent process for producing methylcobalaminhas not yet been established and hence a novel excellent method has beendesired.

Accordingly, an object of the present invention is to provide anindustrially excellent process for producing methylcobalamin, especiallya novel process for producing methylcobalamin, which process does notrequire methyl iodide and purification by column chromatography and isecological. Another object of the present invention is to provide anovel production method which does not invite the formation of amalodorous harmful substance and is ecological, and a process forinhibiting the formation of a malodorous harmful substance in aproduction process of methylcobalamin (V).

DISCLOSURE OF INVENTION

The present invention provides, in an aspect, a process for producingmethylcobalamin (V) which is represented by the following reactionformula including a reduction process and a methylation process.

Reduction process: Cobalamin-CN or Cobalamin-OH→Cobalamin

Methylation process: Cobalamin→Cobalamin-CH₃

Specifically, the present invention provides a process for producingmethylcobalamin (V), comprising the steps of reducing cyanocobalamin (I)or hydroxocobalamin (II) represented by the following formula in thepresence of a reducing agent (III), and then methylating the reductantby adding a water-soluble methylating agent (IV).

-   R²=CN: Cyanocobalamin (I)-   R²=OH: Hydroxocobalamin (II)-   R²=CH₃: Methylcobalamin (V)

In another aspect, the present invention provides a process forproducing methylcobalamin (V), comprising the steps of reducingcyanocobalamin (I) or hydroxocobalamin (II) in an aqueous solution or ahydrous organic solvent in the presence of a reducing agent (III); andthen methylating the reductant by adding a water-soluble methylatingagent (IV).

The present invention further provides a process for producingmethylcobalamin (V), comprising the steps of reducing cyanocobalamin (I)or hydroxocobalamin (II) in an aqueous solution or a hydrous organicsolvent in the presence of a reducing agent (III); methylating thereductant by adding a water-soluble methylating agent (IV); and thenprecipitating the reaction product as crystals or precipitates.

The present invention provides, in yet another aspect, a process forproducing methylcobalamin (V), comprising the steps of reducingcyanocobalamin (I) or hydroxocobalamin (II) in an aqueous solution or ahydrous organic solvent in the presence of a cyanide ion scavenger and areducing agent (III); methylating the reductant by adding awater-soluble methylating agent; and then precipitating the reactionproduct as crystals or precipitates.

In addition, the present invention provides a process for inhibiting theformation of malodorous dimethyl sulfide in a production process ofmethylcobalamin (V) using a trimethylsulfur derivative (VI) as amethylating agent, which comprises the steps of reducing cyanocobalamin(I) or hydroxocobalamin (II) in the presence of a reducing agent (III);and then methylating the reductant by adding trimethylsulfoxoniumiodide, trimethylsulfoxonium bromide and/or trimethylsulfoxoniumchloride.

According to the present invention, the reducing agent and thewater-soluble methylating agent are added separately at differenttimings to the reaction system. Specifically, in the reduction process,the reducing agent is added to the reaction system to thereby convertcyanocobalamin (I) or hydroxocobalamin (II) into a reductant. After thereduction process, the water-soluble methylating agent is added to thereaction system to thereby yield methylcobalamin. In the reductionprocess, whether cyanocobalamin (I) or hydroxocobalamin (II) isconverted into a reductant can be generally verified by whethercyanocobalamin (I) or hydroxocobalamin (II) disappears in a separationanalysis using, for example, high-performance liquid chromatography. Thetermination of hydrogen evolution by action of the reducing agentdemonstrates the completion of the reduction process.

Cyanocobalamin (I), hydroxocobalamin (II) and methylcobalamin (V)relating to the present invention are known natural compounds.

-   Cyanocobalamin (CAS Registry Number: 68-19-9)-   Hydroxocobalamin (CAS Registry Number: 13422-51-0)-   Methylcobalamin (CAS Registry Number: 13422-55-4)

The water-soluble methylating agent (IV) for use in the presentinvention is not specifically limited, as long as it has a solubility inwater at room temperature of 2% or more, and includes, for example,trimethylsulfur derivatives (VI) represented by the following formula.In the formula, X is a halogen atom or a methoxysulfonyloxy group; and nis 0 or 1.

Examples of the trimethylsulfur derivatives (VI) include, but are notlimited to, the following compounds.

-   (1) Trimethylsulfoxonium iodide (CAS Registry Number: 1774-47-6)-   (2) Trimethylsulfonium iodide (CAS Registry Number: 2181-42-2)-   (3) Trimethylsulfoxonium chloride (CAS Registry Number: 5034-06-0)-   (4) Trimethylsulfonium chloride (CAS Registry Number: 3086-29-1)-   (5) Trimethylsulfoxonium bromide (CAS Registry Number: 3084-53-5)-   (6) Trimethylsulfonium bromide (CAS Registry Number: 25596-24-1)-   (7) Trimethylsulfonium methylsulfate (CAS Registry Number:    2181-44-4)

All these compounds are known substances. Among them,trimethylsulfoxonium iodide, trimethylsulfonium iodide,trimethylsulfoxonium chloride, trimethylsulfoxonium bromide andtrimethylsulfonium bromide are available at low cost as reagents orindustrial starting materials. Trimethylsulfonium chloride can be easilyobtained by synthesis according to the method described by B. Byrne etal. in Tetrahedron Lett., 27, 1233, (1986).

Among the trimethylsulfur derivatives (VI), trimethylsulfoxoniumbromide, trimethylsulfonium bromide, trimethylsulfoxonium chloride andtrimethylsulfonium chloride particularly exhibit a high solubility inwater and have a characteristic that the use in a smaller amount yieldshighly pure methylcobalamin in a high yield.

The amount of the trimethylsulfur derivative (VI) is not specificallylimited and is generally from 1.0 to 5 equivalents, preferably from 1.1to 4.5 equivalents, and more preferably from 1.2 to 4 equivalents tocyanocobalamin (I) or hydroxocobalamin (II).

The reducing agent (III) for use in the present invention is notspecifically limited, as long as it can be used in the synthesis ofcyanocobalamin (I) or hydroxocobalamin (II), and includes, for example,sodium borohydride, lithium borohydride, NaBH₃CN (sodiumcyanoborohydride), and Red-Al (sodium bis(2-methoxyethoxy)aluminiumhydride), of which sodium borohydride is preferred.

The amount of the reducing agent (III) is not specifically limited andis generally from 5 to 30 equivalents, preferably from 8 to 25equivalents, and more preferably from 10 to 20 equivalents tocyanocobalamin (I) or hydroxocobalamin (II).

One of features of the present invention is that methylcobalamin havinga high purity equivalent to or higher than products purified by columnchromatography can be conveniently obtained in a high yield in theproduction of methylcobalamin (V) using cyanocobalamin (I) orhydroxocobalamin (II), by sequentially performing a reduction processand a subsequent methylation process stepwise in this order, wherenecessary precipitating a reaction product hardly soluble in water ascrystals or precipitates, and separating and treating the resultingsubstance. In the reduction process, cyanocobalamin (I) orhydroxocobalamin (II) is reduced generally in an aqueous solution or ahydrous organic solvent in the presence of the reducing agent (III). Inthe methylation process, the water-soluble methylating agent (IV) isadded after reduction to thereby methylate the reductant.

The reducing agent and the water-soluble methylating agent are added tothe reaction system in different processes, respectively. Morespecifically, in the reduction process, the reducing agent is added tothereby convert cyanocobalamin (I) or hydroxocobalamin (II) into areductant completely. After the reduction process, the water-solublemethylating agent is added to the reaction system to thereby yieldmethylcobalamin.

Another significant feature of the present invention is that theformation of dimethyl sulfide can be inhibited by sequentiallyperforming the reduction process and the subsequent methylation processstepwise in this order when trimethylsulfoxonium iodide,trimethylsulfoxonium bromide and/or trimethylsulfoxonium chloride amongthe trimethylsulfur derivatives is used as the water-soluble methylatingagent. In the reduction process, cyanocobalamin (I) or hydroxocobalamin(II) is reduced generally in an aqueous solution or a hydrous organicsolvent in the presence of the reducing agent (III). In the methylationprocess, the water-soluble methylating agent (IV) is added afterreduction to thereby methylate the reductant.

Dimethyl sulfide is a malodorous harmful substance and adversely affectsfactory workers and surroundings. Therefore, its emission is severelycontrolled according to the Offensive Odor Control Law.

When the trimethylsulfur derivative serving as the water-solublemethylating agent coexists with the reducing agent in the reactionsystem, the trimethylsulfur derivative is reduced and thereby yieldsdimethyl sulfide that impart burdens on the atmospheric environment.According to the present invention, initially, cyanocobalamin (I) orhydroxocobalamin (II) is reduced with the reducing agent and isconverted into a reductant. After the completion of this process,trimethylsulfoxonium iodide, trimethylsulfoxonium bromide and/ortrimethylsulfoxonium chloride among the trimethylsulfur derivativesserving as the water-soluble methylating agent is added to therebymethylate the reductant. Thus, excess reduction can be prevented and theformation of dimethyl sulfide can be substantially completely inhibited.

When trimethylsulfonium iodide, trimethylsulfonium bromide and/ortrimethylsulfonium chloride is used as the water-soluble methylatingagent, dimethyl sulfide forms but its amount is less than that in a“process for producing methylcobalamin in which the methylating agentand the reducing agent coexist in the reaction system” by sequentiallyperforming the reduction process, wherein cyanocobalamin (I) orhydroxocobalamin (II) is reduced generally in an aqueous solution or ahydrous organic solvent in the presence of the reducing agent (III), andthe subsequent methylation process, wherein the water-solublemethylating agent (IV) is added to a reductant after reduction tothereby methylate the reductant, stepwise in this order. When themethylating agent and the reducing agent coexist in the reaction system,the methylating agent must be added in excess amount for stabilizing thereaction and thereby yields dimethyl sulfide in an amount correspondingto the excess methylating agent. In contrast, “by separatelysequentially performing the reduction process and the subsequentmethylation process” according to the present invention, dimethylsulfide derived from the excess methylating agent is not formed, thusthe amount of formed dimethyl sulfide decreases.

Generally, dimethyl sulfide formed in a reaction process is oftentrapped and removed by 1) an oxidizing agent such as an aqueous solutionof a hypochlorite or 2) an organic solvent such as an aqueous solutionof dimethylformamide. However, the technique 1) is an oxidation reactionusing the oxidizing agent and requires complicated control of complexactions when other components such as hydrogen and hydrogen cyanide inexhausted gas are coexistent. It further requires complicatedinstallation and management of facilities for trapping of the exhaustgas. The technique 2) invites an increased amount of wasted liquid ofthe organic solvent, thus inviting environmental problems and increasedcost caused by the treatment of the waste liquid.

In contrast, the process for producing methylcobalamin and the methodfor inhibiting the formation of dimethyl sulfide according to thepresent invention control the formation of dimethyl sulfide itself, donot require additional extra facilities and treatments and are veryconvenient and useful methods.

The production method according to the present invention enables theproduction of highly pure methylcobalamin in a high yield using no metalion or using only a small amount thereof as a cyanide ion scavenger, andthe method exhibits an extremely excellent effect in view that noproblem arises at removal of metal ion products, which is difficult tofilter, from the system.

When methyl iodide is used as a methylating agent, ferrous sulfate isgenerally often used as a cyanide ion scavenger in combination withmethyl iodide. The amount of ferrous sulfate in this case must be 30% byweight or more relative to cyanocobalamin (I) or hydroxocobalamin (II).

However, the present invention enables the production of highly puremethylcobalamin in a high yields because methylation proceeds even whenno ferrous sulfate is used as a cyanide ion scavenger.

When a small amount of ferrous sulfate is used as a cyanide ionscavenger, the reaction proceeds at a higher rate, and highly puremethylcobalamin can be obtained in a high yield even by the sameaftertreatment procedure as in the case where no ferrous sulfate isused. Likewise, by using a small amount of cobalt chloride, themethylation reaction proceeds with high selectivity to thereby inhibitthe formation of impurities, and highly pure methylcobalamin can also beobtained in a high yield.

Examples of the cyanide ion scavenger for use in the present invention,metals or metal salts such as ferrous sulfate, iron powder, Mohr's salt,ferrous chloride, cobalt chloride, nickel chloride or zinc chloride maybe proposed. Among them, ferrous sulfate and/or cobalt chloride isparticularly preferred. Each of these metals and metal salts can be usedalone or in combination.

The amount of the cyanide ion scavenger can be small and is generallyfrom 1 to 30% by weight and preferably from 1 to 10% by weight relativeto cyanocobalamin (I) or hydroxocobalamin (II).

The use of a reaction solvent is not specifically limited, and thereaction solvent, if used, is not specifically limited as long as it isinert to cyanocobalamin (I), hydroxocobalamin (II), trimethylsulfurderivative (VI) and methylcobalamin (V). The reaction solvent isgenerally an aqueous solution or a hydrous organic solvent. The organicsolvent is preferably one soluble in water, lower alcohols such asmethanol, ethanol, propanol, isopropanol, butanol, isobutanol,sec-butanol or t-butanol; esters such as methyl formate, ethyl formate,methyl acetate, ethyl acetate or isopropyl acetate; ketones such asacetone, 2-butanone or 3-methyl-2-butanone; cyclic ethers such as THF ordioxane; as well as acetonitrile, DMF, DMSO, pyridine, and mixtures ofthese organic solvents.

The reaction temperature in the reduction process and the methylationprocess in the present invention is not specifically limited and isgenerally from 0° C. to 90° C., preferably from 10° C. to 70° C., andmore preferably from 15° C. to 50° C.

The reactions in the reduction process and the methylation process inthe present invention are preferably performed under flow of an inertgas such as nitrogen gas and/or in the dark (under red light). However,the reaction procedures are not limited thereto.

The present invention can provide an industrially excellent process forproducing methylcobalamin. It can also provide a novel industriallyexcellent process for producing methylcobalamin, which method does notinvite the formation of a malodorous harmful substance and isecological, and a process for inhibiting the formation of a malodorousharmful substance in a production process of methylcobalamin (V).Examples of the advantages of the present invention will be describedbelow. Inhibitory effect of present invention on formation of malodorousharmful substance

The method of the present invention comprises performing reactions of areduction process and of a subsequent methylation process stepwise inthis order.

In Examples 1 to 3 (methylating agent: trimethylsulfoxonium bromide)according to the present invention, the concentration of dimethylsulfide in a reaction pot was measured in the following manner everyhour from immediately after the completion of the dropwise addition ofan aqueous solution of sodium borohydride. Specifically, dimethylsulfide was introduced from the reaction pot via a glass tube to a 50%DMF aqueous solution as an absorbent, was subjected to gas absorption bygas-liquid bubbling, and the concentration of collected dimethyl sulfidein the absorbent was determined by capillary gas chromatography.(Capillary gas chromatography was performed using GC HP 6890manufactured by Agilent, and a DB-624 column. After retaining at 50° C.for 10 minutes, the temperature was elevated to 200° C. at a rate of 15°C. per minute. The gas absorption temperature was 100° C., the detectiontemperature was 215° C., and the injection amount was 1 μl. At the sametime, a sensory test of odor was performed.

As a control experiment, the same procedure was repeated every hour fromimmediately after the completion of dropwise addition of an aqueoussolution of sodium borohydride in the following “process for producingmethylcobalamin in which a trimethylsulfur derivative as a water-solublemethylating agent coexists with a reducing agent in the reactionsystem”.

Control Experiment

To 260 ml of ion-exchanged water were added 20 g of cyanocobalamin, 7.66g of trimethylsulfoxonium bromide, 1.4 g of cobalt chloride hexahydrate,and 15 ml of 2-butanone. After replacing the atmosphere of the systemwith nitrogen gas, the mixture was heated in a water bath, to which anaqueous solution of sodium borohydride (8 g/40 ml) was added dropwise atthe inner temperature of 40° C. over 90 minutes under stirring. Afterstirring for further 3 hours as it was, the mixture was stirred at abath temperature of 15° C. overnight. The resulting precipitates werecollected by filtration and dried, to give a crude product of the titlecompound. To the crude product was added a 50% acetone aqueous solution,the mixture was heated at 35° C., was adjusted to pH 7.0 withconcentrated hydrochloric acid, followed by dropwise addition of acetoneand stirring overnight. The precipitated crystals were collected byfiltration and were dried.

The concentrations of formed dimethyl sulfide in each Example are shownin Table 1.

TABLE 1 the intervals after immediately the dropwise after the additionof dropwise sodium borohydride addition 1 hour 2 hour 5 hour Example 1  0 ppm   0 ppm  0 ppm 0 ppm odorless odorless odorless odorless Example2   0 ppm   0 ppm  0 ppm 0 ppm odorless odorless odorless odorlessExample 3   0 ppm   0 ppm  0 ppm 0 ppm odorless odorless odorlessodorless Control Example 1 62.4 ppm 1250 ppm 697 ppm malodorousmalodorous malodorous the upper line: concentration of dimethyl sulfidethe lower line: result of the sensory test of odor

In Examples 1 to 3 (methylating agent: trimethylsulfoxonium bromide), nomalodor was perceived in the sensory test and no dimethyl sulfide wasdetected in gas chromatography 1 hr, 2 hr, 3 hr, and 5 hr after theaddition of sodium borohydride. In contrast, in the control example inwhich the water-soluble methylating agent and the reducing agent were incoexistence, malodor was perceived, and dimethyl sulfide in a highconcentration was detected at least 3 hr after the addition.

These results show that the production method of methylcobalaminaccording to the present invention, especially the production method “inwhich cyanocobalamin (I) or hydroxocobalamin (II) is reduced in thepresence of the reducing agent (III), and trimethylsulfoxonium iodide,trimethylsulfoxonium bromide and/or trimethylsulfoxonium chloride isadded after reduction to thereby methylate the reductant”, does notinvite and can effectively inhibit the formation of a malodorous harmfulsubstance.

EXAMPLES

The present invention will be illustrated in further detail withreference to Examples below, which are not intended to limit the scopeof the invention.

Example 1 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 65 ml of ion-exchanged water were added 5 g of cyanocobalamin, 0.35 gof cobalt chloride hexahydrate, and 3.75 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (2 g/10 ml) was added dropwise under stirring at abath temperature of 38° C. over 60 minutes. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (1.9 g/10 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 85%. Physical properties of the resultingmethylcobalamin:

-   -   In a hydrochloric acid buffer (pH 2.0), UVmax was detected at        264–266, 303–307 and 459–462 nm.    -   In a phosphate buffer (pH 7.0), UVmax was detected at 266–269,        341–344 and 520–524 nm.    -   Referential values of UVmax (Merck Index, 12th edition)        -   (0.1 N—HCl): 264, 304 and 462 nm        -   (pH 7): 266, 342 and 522 nm

Example 2 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 260 ml of ion-exchanged water were added 20 g of cyanocobalamin, 1.4g of cobalt chloride hexahydrate, and 15 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (8 g/40 ml) was added dropwise under stirring at theinternal temperature of 40° C. over 70 minutes. After stirring forfurther 30 minutes as it was, an aqueous solution oftrimethylsulfoxonium bromide (7.66 g/40 ml) was further added theretoover 30 minutes. The mixture was stirred for further 3 hours as it was,followed by stirring overnight at a bath temperature of 15° C. Theresulting precipitates were collected by filtration and dried to give acrude product of the title compound. To the crude product was added a50% acetone aqueous solution. After heating at 35° C., the mixture wasadjusted to pH 7.0 with concentrated hydrochloric acid. Then, acetonewas added dropwise thereinto and the mixture was stirred overnight. Theprecipitated crystals were collected by filtration and dried, to givethe title compound in a yield of 85%.

Example 3 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 30 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 40° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 88%.

Example 4 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 30 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 40° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 87%.

Example 5 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 30 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 50° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 88%.

Example 6 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 30 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 50° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 87%.

Example 7 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 3 0 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 30° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 85%.

Example 8 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 390 ml of ion-exchanged water were added 3 0 g of cyanocobalamin, 2.1g of cobalt chloride hexahydrate, and 22.5 ml of 2-butanone. Whileblowing nitrogen gas at the flow rate of 15 ml/min. in to the system,the mixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (12 g/60 ml) was added dropwise under stirring at theinternal temperature of 30° C. over 2 hours. After stirring for further30 minutes as it was, an aqueous solution of trimethylsulfoxoniumbromide (11.5 g/60 ml) was further added thereto over 30 minutes. Themixture was stirred for further 3 hours as it was, followed by stirringovernight at a bath temperature of 15° C. The resulting precipitateswere collected by filtration and dried to give a crude product of thetitle compound. To the crude product was added a 50% acetone aqueoussolution. After heating at 35° C., the mixture was adjusted to pH 7.0with concentrated hydrochloric acid. Then, acetone was added dropwisethereinto and the mixture was stirred overnight. The precipitatedcrystals were collected by filtration and dried, to give the titlecompound in a yield of 86%.

Example 9 Synthesis of Methylcobalamin

All the procedures in the present example were performed in the dark(under red light).

To 13 L of ion-exchanged water were added 1 Kg of cyanocobalamin, 70 gof cobalt chloride hexahydrate, and 750 ml of 2-butanone. Afterreplacing the inside atmosphere of the system with nitrogen gas, themixture was heated in a water bath, to which an aqueous solution ofsodium borohydride (400 g/2 L) was added dropwise while keeping andstirring at the internal temperature of 35° C.±5° C. over 120 minutes.An aqueous solution of trimethylsulfoxonium bromide (383 g/2 L) wasfurther added thereto over 30 minutes. The mixture was stirred forfurther 3 hours as it was, followed by stirring overnight at a bathtemperature of 15° C. The resulting precipitates were collected byfiltration and dried to give a crude product of the title compound. Tothe crude product was added a 50% acetone aqueous solution. Afterheating at 35° C., the mixture was adjusted to pH 7.0 with concentratedhydrochloric acid. Then, acetone was added dropwise thereinto and themixture was stirred overnight. The precipitated crystals were collectedby filtration and dried, to give the title compound in a yield of 87%.

1. A two-step process for producing methylcobalamin (V), comprising thesequential steps of A) and B): A) reducing cyanocobalamin (I) orhydroxocobalamin (II) represented by the following formula in thepresence of a reducing agent (III) that is sodium borohydride, and B)methylating the reductant by adding a water-soluble methylating agent(IV)

R²=CN: Cyanocobalamin (I) R²=OH: Hydroxocobalamin (II) R²=CH:Methylcobalamin (V) wherein the water-soluble methylating agent (IV) isa trimethylsulfur derivative (VI) represented by the following formula:

wherein X is a halogen atom or a methoxysulfonyloxy group, and n is 0 or1; and wherein the amount of the trimethylsulfur derivative (VI) addedis from 1.0 to 5.0 equivalents to cyanocobalamin (I) or hydroxocobalamin(II).
 2. A two-step process for producing methylcobalamin (V),comprising the sequential steps of A) and B): A) reducing cyanocobalamin(I) or hydroxocobalamin (II) in an aqueous solution or a hydrous organicsolvent in the presence of a reducing agent (III) that is sodiumborohydride; and B) methylating the reductant by adding atrimethylsulfur derivative (VI) represented by the following formula:

wherein X is a halogen atom or a methoxysulfonyloxy group, and n is 0 or1; and wherein the amount of the trimethylsulfur derivative (VI) addedis from 1.0 to 5.0 equivalents to cyanocobalamin (I) or hydroxocobalamin(II).
 3. A multi-step process for producing methylcobalamin (V),comprising the sequential steps of A) to C): A) reducing cyanocobalamin(I) or hydroxocobalamin (II) in an aqueous solution or a hydrous organicsolvent in the presence of a reducing agent (III) that is sodiumborohydride; B) methylating the reductant by adding a trimethylsulfurderivative (VI) represented by the following formula:

wherein X is a halogen atom or a methoxysulfonyloxy group, and n is 0 or1; and C) precipitating the reaction product as crystals orprecipitates; and wherein the amount of the trimethylsulfur derivative(VI) added is from 1.0 to 5.0 equivalents to cyanocobalamin (I) orhydroxocobalamin (II).
 4. A multi-step process for producingmethylcobalamin (V)1 comprising the sequential steps of A) to C): A)reducing cyanocobalamin (I) or hydroxocobalamin (II) in an aqueoussolution or a hydrous organic solvent in the presence of a cyanide ionscavenger and a reducing agent (III) that is sodium borohydride; B)methylating the reductant by adding a trimethylsulfur derivative (VI)represented by the following formula:

wherein X is a halogen atom or a methoxysulfonyloxy group, and n is 0 or1; and C) precipitating the reaction product as crystals orprecipitates; and wherein the amount of the trimethylsulfur derivative(VI) added is from 1.0 to 5.0 equivalents to cyanocobalamin (I) orhydroxocobalamin (II).
 5. The process for producing methylcobalamin (V)according to claim 1, 2, 3 or 4, wherein the trimethylsulfur derivative(VI) is trimethylsulfoxonium iodide, trimethylsulfonium iodide,trimethylsulfoxonium bromide, trimethylsulfonium bromide,trimethylsulfoxonium chloride and/or trimethylsulfonium chloride.
 6. Theprocess for producing methylcobalarnin (V) according to claim 4, whereinthe cyanide ion scavenger is ferrous sulfate and/or cobalt chloride. 7.The process for producing methylcobalamin (V) according to claim 4 or 6,wherein the amount of the cyanide ion scavenger is from 1 to 30% byweight relative to cyanocobalamin (I) or hydroxocobalamin (II).